Structural Chromosomal Abnormalities Flashcards
State the 6 major types of structural abnormalities that can arise?
- Translocations: Reciprocal, Robertsonian
- Inversion
- Deletion
- Duplication
- Rings
- Isochromosomes
What is translocation?
- Exchange of two segments between non-homologous chromosomes (not paired)
- Occurs via inappropriate non-homologus end joining (NHEJ)
How does NHEJ translocation arise?
- DNA repair mechanism
- Common for double strands break to occur in chromosomes causing chunks to split off
- NHEJ will rejoin the chunk back to the original chromosome
- Inappropriate NHEJ will join the segment to another chromosome and vice versa forming derivative chromosomes
What is the common result of inappropriate non-homologous end joining?
- Carriers of balanced translocation (no. clinical effect)
- Forms 2 derivative chromosomes + copies of both normal chromosome (due to HC)
- No net gain or loss of genetic materal (any chromosome and size fragment), just material is in wrong place
Give an example of a carrier of balanced translocation which have adverse effects?
- One that forms the philadelphia chromosome (Cr 9- ABL +Cr 22-BCR)
- Learn below VD
- Can result in fusion gene leading to leukaemia
- Diagnosed via G-banding
How are unbalanced individuals produced?
When they have < or > of a particular region of a chromosome
What is reciprocal translocation and state the common problem?
- Reciprocal translocation is a form of gene rearrangement where portions of two chromosomes are simply exchanged with no net loss of genetic information
- Common problem arise when the derivative chromosomes align with their homologue as some may contain 2 copies
State the consequence of the reciprocal translocations in this picture? VD important
- Individual has a mixture of trisomic + monosomic with respect to chromosome
- 3 copies of purple so trisomic
- Tetravalent formed (4 chromosomes)
State the common results of unbalanced reciprocal translocation (3)?
- Many lead to miscarriage (hence why a woman with a high number of unexplained miscarriages should be screened for a balanced translocation)
- Learning difficulties, physical disabilities
- Tend to be specific to each individual so exact risks and clinical features vary
What is robertsonian translocation?
- Two acrocentric chromosomes join via q arms (long) at centromere with the loss of p/satellites arms (short)
- Balanced carrier has 45 chromosomes - with one tetravalent formed
- If 46 chromosomes present including Robertsonian then must be unbalanced - as you’ll lose a chromosome where the 2 A chr. combine together.
- p arms encode rRNA (multiple copies so not deleterious to lose some)
- Only involved in acrocentric chromosome
- P arms of both chromosomes are removed, Q arm of both chromosomes combine to form robertsonian translocation
State which chromosomes are common for robertsonian translocations
to occur and what can it potentially lead to?
- Robertsonian translocations 13;14 and 14;21 relatively common.
- 21;21 translocation leads to 100% risk of Down syndrome in fetus
Give the possible gemetes and outcomes that can be formed from this
VD
What are the 5 main outcomes of translocations?
- Very difficult to predict
- Only have approximate probability of producing possible gametes
- Some unbalanced outcomes may lead to spontaneous abortion of conceptus so early that not seen as problem
- Some unbalanced outcomes may lead to miscarriage later on and present clinically
- Some may result in live-born baby with various problems
State the two types of deletion and the effects?
- Deletion may be terminal (loss of telomere) or interstitial (middle segment)
- Causes a region of monosomy - copies of particular region in only one chromosome
- Haploinsufficiency of some genes - The situation that occurs when one copy of a gene is inactivated or deleted and the remaining functional copy of the gene is not adequate to produce the needed gene product to preserve normal function
- Contiguous gene syndrome (multiple, unrelated clinical features): Due to deletion at different chromosomes potentially
- Phenotype is specific for size and place on deletion
- Gross deletions seen on metaphase spread on G-banded karyotype
State how microdeletions can be seen?
- Many patients had no abnormality visible on metaphase spread
- High resolution banding, FISH and now CGH showed ‘micro’ deletions
- Only a few genes may be lost or gained
State how gross deletions can be seen?
Metaphase spread on G-banded karyotype
State 4 examples of diseases that can arise from microdeletions including the chromosomes?
- Velocardiofacial (DiGeorge), 22q11
- Wolf-Hirschhorn, 4p16
- Williams, 7q11
- Smith-Magenis, 17p11
State 3 examples of diseases that can arise from interstital deletion?
- Prader-Willi
- DiGeorge Syndrome
- Cri du chat
What mechanism results in (micro) deletions and (micro) duplications?
- Unequal crossing over/ non-allelic homologous recombination
- Unequal alignment during crossing over
- Potential deletion or duplication of genes occuring
State prenatal (3) and postnatal (2) sources for detecting structural abnormalities?
- Prenatal
- Amniocentesis - sample of amniotic fluid for fetal DNA
- Chorionic villus sampling: Piece of placenta taken for access to fetal DNA
- Cell-free fetal DNA: Not invasive, above two are
- Postnatal: Blood, Saliva
Describe G-banding
- G = Giemsa
- Main Purpose: How does karyotype of patient differ from expected?
- Looks for aneuploidies, translocations & very large deletions
Describe the point of the G banding technique
- Key points of technique:
- Uses a chemical stain (Giemsa)
- Uses metaphase chromosomes - DNA is densely packed
- Takes several days at least
Why are bands produced in G-banding?
- Chromatin - 2 different sorts: euchromatin & heterochromatin
- Euchromatin = GC-rich; loosely packed; genes active = Light
- Heterochromatin = AT-rich; tightly packed; genes inactive = Dark
- Stain differently
Describe the process of staining in G banding
- 5 mL venous blood -> Add phytomagglutinin and culture medium -> culture at 37° for 3 days -> Add Colchicine and hypotonic saline -> Cells fixed -> Spread cells into slide by dropping -> Digest with trypsin and stain with giemsa -> Analyse metaphase spread
- Quite a slow process
Describe FISH
- Fluorescent in situ hybridisation
- Main purpose: How does karyotype of patient differ from expected? Looks for aneuploidies, translocations & large deletions
- Cultured cells, metaphase spread (chromosomes) used
Describe the process of the FISH technique?
- Fluorescent probe - for specific parts of the genome
- Denature probe and target DNA
- Mix probe and target DNA
- Probe binds to target - visualise using fluorescent
- Takes several days
What is hybridisation?
Hybridisation = single stranded nucleic acid binds to a new single stranded nucleic acid (DNA/DNA or DNA/RNA)
What is a probe?
- A single stranded DNA (or RNA) molecule - complementary to target DNA
- Typically 20 - 1000 bases in length
- Labelled with a fluorescent or luminescent molecule (less commonly a radioactive isotope)
- In some techniques thousands or millions of probes are used simultaneously
State two diseases that FISH can be used for?
- Cri-du-chat (5p-syndrome)
- 22q deletion syndrome
Describe the purpose of the array CHG technique
- Array comparative genomic hybridisation
- Main purpose: How many copies of a particular genomic region does the patient have +
- Detection of sub-microscopic chromosomal abnormalities:
microdeletions and microduplications
Describe the process of the Array CGH technique
- Uses fluorescent probes to differentiate between patient and control
- Patient DNA is labelled green and control DNA is labelled red.
- Uses extracted DNA
Describe the purpose of the Qf-PCR technique
- Quantitive fluorescence polymerase chain reaction
- Main purpose: How many copies of a chromosome does the patient have?
- Looks for aneuploidies (trisomies 13,18 and 21)
Describe the process of the QF-PCR technique
- Uses fluorescent probes for SPECIFIC microsatellite markers on SPECIFIC chromosomes
- Uses extracted DNA
- Quick (~48hrs)
- Need to know what MS you’re looking for
What are microsatellites?
- Short repeated sequences
- Number of repeats varies between individuals
- Total length of microsatellite sequence varies between individuals
- Microsatellites are distributed across the whole genome - most are not within genes.
How do you detect microsatellites in Qf-PCR?
- Isolate DNA from individual
- Design primers specific to flanking sequences
- PCR amplification
- Gel electrophoresis
- PCR amplification of microsatellite region
- Genotype size of fragments on gel-based system
a. Homozygotes = single product of specific size
b. Heterozygotes = two different sized products
What is PCR?
Exponential amplification of a DNA fragment of known sequence
State the key components of the PCR reaction?
- Components of the PC reaction:
- Template - DNA to amplify
- Primers - Short pieces of SSDNA (15-30bp)
- Polymerase - thermostable enzyme (Taq)
- Nucleotides - single base mixture (dNTPs)
- Buffer - To maintain pH
- MCI2 - Essential for polymerase activity
State the stages of the PCR cycle?
- Denaturation - heat and separate DNA strands at 94 degrees
- Annealing - Primers anneal with template (50-65 degrees approx. 60)
- Extension - DNA polymerase extends strand from primer (72 degrees)
- PC exponential amplification occurs until components of reaction run out causing the reaction to slow down and stop
Describe the graph and results of Qf-PCR on chromosome 21?
- Perform PC using primers for microsatellite known to be on chromosome 21 (if testing for Down’s)
- Should be two copies of microsatellite (one from mother, one from father, like any other autosomal locus, gene, whatever)
- If homozygous, there will be a single peak of high signal
- If heterozygous, there will be two peaks of similar, lower signal
What is NIPT and NGS?
- NIPT = Non-invasive pre-natal testing
- NGS = Next generation sequencing
What are the purposes of this technique?
- Main purpose: ‘High chance’ indicator for invasive test of aneuplodies
- Uses cell free fetal (extracted) DNA from maternal blood sample
- Utilises next generation sequencing
- Screening not diagnostic
